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Lab 6: Introduction to the Microscope

BIO105 Introduction to Biology

What are going to learn about?

▪ Understand the basic Care of Microscope

▪ Understand the Inversion of Image

▪ Be able to Focus the Microscope ( Low Power)

▪ Understand :

▪ Total Magnification

▪ Diameter field of view

▪ Depth of Focus

▪ Observing a prepared slide

Learning Objectives

What can you see with a light microscope?

1000 millimeters (mm) = 1 meter

(m)

1000 micrometers (µm or mcm) = 1

millimeter (mm)

1000 nanometers (nm) = 1

micrometer (mcm)

A B

Carrying a Microscope

• Return the lowest power

objective in place

• Wrap the cord around the

base

• Cord is tight

Storing The Microscope

Basic Care of Microscope

How to carry

• Both hands

• Make sure you remove slides before returning to cabinet.

Cleaning

• Clean eyepieces with lens tissue

• Both before and after use.

View and Focus

• Always view using scanning power (4x)

• Focus on low power

Putting Away

• Wrap cord tightly around and neatly

• Place back in cabinet.

Purpose of the Microscope

Magnification The degree to which the image of a specimen is enlarged.

Resolution The ability to distinguish two objects as separate and

distinct.

Contrast

The ability to see specimen detail against its background.

Stains and dyes are added to sections of biological

specimens to increase contrast.

Microscope Parts

A. Ocular

B. Body tube

C. Stage clip

D. Revolving nose piece

E. Objective

F. Arm

G. Stage

H. Diaphragm

I. Lever to move stage clip

J. Course adjustment

K. Fine adjustment

L. Light source

M. Base

Using the microscope

• Always observe using the LOWEST POWER objective

first.

• Focus using the COARSE ADJUSTMENT KNOB to

bring the object into focus. Bring the object into sharp

focus by using the fine adjustment knob.

• Focus, and then move to a higher power objective, if

needed.

• Use only the FINE ADJUSTMENT KNOB when using

the HIGHEST (longest) POWER OBJECTIVE.

• Keep both eyes open to reduce eyestrain.

• Determine total magnification of the object by

multiplying the power of the ocular (10x) the power by

the power of the objective.

Preparing a slide

• Using a pipet or dropper, add a drop of water or

another solvent to a clean microscope slide.

Then, place the specimen in the water.

• Place the edge of a coverslip on the slide so that

it touches the edge of the water.

• Slowly lower the coverslip to prevent the

formation of air bubbles.

A. Appearance of objects

1. Inverted and reversed (upside-down & backwards)

2. If an "e" is placed in the stage in its normal position, it will appear as upside down &

inverted 3. Only a thin layer of the specimen is in focus at any level (depth of focus).

" ".

1. Focusing the Microscope

Start with low focus

▪ Turn nosepiece to lowest power(4X)

▪ 4X – Start with Scanning Power

Microscope Slide

Upside down & inverted

Movement of specimens

1. Actual movement is opposite to appeared direction of movement.

2. If an organism is actually moving (  ), it will appear to be moving (  ).

100x40x

Lens: Inversion of Image

Move the slide to the right while viewing through the eyepiece. Which direction does the word “roe” appear to move? Opposite direction

Slide #1: “roe”

Inversion of Image

100x40x

Move the slide towards you. Which direction does the word “roe” appear to move? away

▪ Place slide on microscope and secure

▪ Start with scanning objective - center and focus. See any thing unusual?

▪ Change to low power and draw

▪ Change to high power and draw

Depth of View

▪ Notice that as you increase magnification you see more detail of less of the image.

▪ Higher magnification lenses look at smaller fields of view

▪ You also see layer of the image that you can focus through

▪ More of less

Ocular Lens

❖Ocular lens magnifies the specimen 10x.

❖You will always be looking through the ocular and objective lens simultaneously, so multiply ocular magnification x objective power to calculate the Total Magnification (xTM).

❖Rotary nosepiece of your microscope has four objective lenses attached

3. Focusing the Microscope- Low Power

Magnification Eyepiece (Ocular)

What you look through May have pointer inside 10x

Rotating nosepiece has 3 objectives Scanning (short and red) 4x Low (medium and yellow) 10x High (long and blue) 40 x

Total Magnification = Ocular X Objective

Eyepiece 10x

Scanning 4x = 40x total Low 10x = 100x total High 40x = 400x total

5. Total Magnification

▪ Calculate the magnification for the combined eyepiece and objective lens.

▪ Multiply ocular magnification times objective magnification.

Eyepiece Lens

Magnification

Objective

Lens Power Total Magnification

10X 4X (Scanning) 40x

10X 10X (Low Power) 100x

10X 40X (High Power) 400x

10X 100X (Oil Immersion) 1000x

When changing objectives from scanning power to lower power to high power, the following changes will occur:

a. the size of the field of view decreases.

b. the field of view is darker.

c. the size of the image increases.

d. the resolution (ability to separate small details) increases.

e. the working distance (distance between coverslip & objective) decreases.

f. the depth of focus (thickness of the specimen which may be seen in focus) is reduced.

Field of view

Scanning power (50X) is about 3.0 to 3.2 mm and low power (100X) is about 1.5 to 1.6 mm. Sizes will vary with changes in magnification and manufacturer

Convert the measurement in millimeters to micrometers by multiplying by 1000.

Samples: low power field 1.6 mm X µm/mm = 1600 µm or mcm

scanning power field 3.2 mm X 1000 µm/mm = 3200 µm or mcm

The diameter of the high power field is less than one millimeter.

It can be calculated

from the diameter of the low power field by using the following

formula:

high power field diameter = low power magnification

lower power field diameter high power magnification

Sample:

h.p. field = 100 to h.p. field = 100

X 1600 = 400 µm or mcm

1600 µm 400 400

Focusing

▪ IV. Focusing

▪ Place slide on stage and secure

▪ Use scanning objective and try to center the crossed threads then focus ▪ Use course focus (large knob) first

▪ Fine focus (small knob) second

▪ Which way does the slide move if you push it left? Push right?

▪ What affect does changing the diaphragm have on the image?

▪ Change to low power ▪ Notice any change?

▪ Draw

Slide #2 - letter “colored thread”

▪ Prepared slide of crossed threads

Resolving Power

▪ A measure of the clarity of an image.

Eyepiece Lens

Magnification

Objective

Lens Power Total Magnification

10X 4X (Scanning) 40x

10X 10X (Low Power) 100x

10X 40X (High Power) 400x

10X 100X (Oil Immersion) 1000x

Closure

▪ Complete post lab questions

▪ Turn in lab

Clean Up